Bottom Line:
In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used.The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the -4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously.In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs.

Background: Inhibitors of pancreatic alpha-amylase are potential drugs to treat diabetes and obesity. In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used. The accuracy of models is limited, but these tools are nonetheless valuable. In vitro models could be used in large screenings involving thousands of chemicals that are tested to find potential lead compounds. In vivo models are still used as preliminary mean of testing compounds behavior in the whole organism. In the case of alpha-amylase inhibitors, both rats and rabbits could be chosen as in vivo models. The question was which animal could present more accuracy with regard to its pancreatic alpha-amylase.

Results: As there is no crystal structure of these enzymes, a molecular modeling study was done in order to compare the rabbit and rat enzymes with the human one. The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the -4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously.

Conclusion: Molecular modeling tools could be used to choose the most suitable model enzyme that would help to identify new enzyme inhibitors. In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs.

Figure 3: a. Coordination state of the calcium ion as found in the three-dimensional structures of human enzyme, as well as in rabbit and rat enzymes.b. Interactions observed between the chloride ion of human, rabbit, and rat enzymes. The ligand interaction module of MOE.2009.10 has been used to obtain the two-dimensional schematic representation of the residues positioned in the vicinity of the ions. In order to provide a better feeling of the three-dimensional arrangement of the shown residues in the case of chloride, this site is shown in rabbit enzyme, with chloride ion shown as a sphere, and nearby amino acids as ball and sticks. Colour codes of amino acids are as follows: basic residues in pink with blue border, acidic residues in pink with red border, polar residues in pink, hydrophobic (greasy) residues in green.

Mentions:
The superimposed minimized backbone structures of the three enzymes are shown in Figure 2, where the three domains of the enzymes (A-C) are indicated. Positioning of the calcium and chloride ions are related to the human one, and could be assumed to be similar in RPA and RABPA. The nearby residues interacting with these ions are shown in Figure 3a and b for comparison means and indicate no major difference between the three enzymes, a fact that is a further indication of the biological plausibility of the models. The calcium ion of HPA makes its interactions with the residues of domain B, with the exception of His201 which belongs to Domain A [20]. This ion is considered to be “a central organizing structural feature of Domain B” [24], is needed for alpha-amylase catalytic activity, and its binding pocket is essential to maintain the fold stability [25]. The chloride ion was previously thought to be a feature of mammalian amylase, and shown to possess activating property [26]. In a thorough study to characterize the chloride binding pocket of alpha-amylases, the anion was found to be coordinated by one arginine, one arginine/lysine and an asparagine residue throughout chloride-dependent amylases, with R337 (PPA numbering) being a key feature for the anion binding [27].

Figure 3: a. Coordination state of the calcium ion as found in the three-dimensional structures of human enzyme, as well as in rabbit and rat enzymes.b. Interactions observed between the chloride ion of human, rabbit, and rat enzymes. The ligand interaction module of MOE.2009.10 has been used to obtain the two-dimensional schematic representation of the residues positioned in the vicinity of the ions. In order to provide a better feeling of the three-dimensional arrangement of the shown residues in the case of chloride, this site is shown in rabbit enzyme, with chloride ion shown as a sphere, and nearby amino acids as ball and sticks. Colour codes of amino acids are as follows: basic residues in pink with blue border, acidic residues in pink with red border, polar residues in pink, hydrophobic (greasy) residues in green.

Mentions:
The superimposed minimized backbone structures of the three enzymes are shown in Figure 2, where the three domains of the enzymes (A-C) are indicated. Positioning of the calcium and chloride ions are related to the human one, and could be assumed to be similar in RPA and RABPA. The nearby residues interacting with these ions are shown in Figure 3a and b for comparison means and indicate no major difference between the three enzymes, a fact that is a further indication of the biological plausibility of the models. The calcium ion of HPA makes its interactions with the residues of domain B, with the exception of His201 which belongs to Domain A [20]. This ion is considered to be “a central organizing structural feature of Domain B” [24], is needed for alpha-amylase catalytic activity, and its binding pocket is essential to maintain the fold stability [25]. The chloride ion was previously thought to be a feature of mammalian amylase, and shown to possess activating property [26]. In a thorough study to characterize the chloride binding pocket of alpha-amylases, the anion was found to be coordinated by one arginine, one arginine/lysine and an asparagine residue throughout chloride-dependent amylases, with R337 (PPA numbering) being a key feature for the anion binding [27].

Bottom Line:
In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used.The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the -4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously.In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs.

Background: Inhibitors of pancreatic alpha-amylase are potential drugs to treat diabetes and obesity. In order to find compounds that would be effective amylase inhibitors, in vitro and in vivo models are usually used. The accuracy of models is limited, but these tools are nonetheless valuable. In vitro models could be used in large screenings involving thousands of chemicals that are tested to find potential lead compounds. In vivo models are still used as preliminary mean of testing compounds behavior in the whole organism. In the case of alpha-amylase inhibitors, both rats and rabbits could be chosen as in vivo models. The question was which animal could present more accuracy with regard to its pancreatic alpha-amylase.

Results: As there is no crystal structure of these enzymes, a molecular modeling study was done in order to compare the rabbit and rat enzymes with the human one. The overall result is that rabbit enzyme could probably be a better choice in this regard, but in the case of large ligands, which could make putative interactions with the -4 subsite of pancreatic alpha-amylase, interpretation of results should be made cautiously.

Conclusion: Molecular modeling tools could be used to choose the most suitable model enzyme that would help to identify new enzyme inhibitors. In the case of alpha-amylase, three-dimensional structures of animal enzymes show differences with the human one which should be taken into account when testing potential new drugs.